1. Field of the Invention;
This invention relates to a substance-conjugated complement component C1q and a process for preparing the same, and a method for measurement, using the substance-conjugated complement component C1q. More particularly, it relates to a complement component C1q conjugated with various substances including markers and cell function regulating substances and a process for preparing the same, and also to a process for measurement, using such a substance-conjugated complement component C1q.
2. Related Art Statement;
It has hitherto been known to utilize the complement fixation reaction for the measurement or determination of antibodies in blood serum and antigens, such as microorganisms, phisiologically active substances and chemicals. This known method makes use of the serial reactions wherein complement components C1 to C9 are bound successively to an antibody specifically bound to an antigen. In detail, this known method comprises the step of adding an excess amount of complement components to the formed antigen-antibody complex, the step of determining an amount of residual complement components through the hemolysis, and the step of determining the amount of fixed complement components from the degree of hemolysis. The quantity of the antigen or antibody is then estimated from the results of the amount of the fixed complement components. In the hemolysis, complement components act on the sensitized erythrocytes including sheep red blood cells and anti-sheep red blood cell antisera so that the complement components may be determined while using the hemolysis of the sheep red blood cells as the index. However, practical determination operation of the hemolysis is extremely complex and needs high level skill and knowledge. In addition, this known method has a relatively low sensitivity and requires two days for the determination operation.
Various methods have been proposed to overcome the aforementioned disadvantages of the known method as described in the preceding paragraph. For example, Japanese Patent Laid-Open Publication No. 43498/1980 discloses one of such methods. In the method proposed by the antecedent Publication referred to above, an antibody which binds, as an antigen, a complement component being bound to another antibody is labelled with an enzyme, and the amount of the thus labelled antibody is determined by the enzymatic activity thereof. This method is, therefore, one of the so-called enzyme-labelled antibody techniques. However, this method involves two step reactions, since a labelled antibody which binds, as an antigen, a complement component must be used. Accordingly, rinsing operations are required after each of the reactions, leading to increase in labor and time. In fact, this determination method costs much time as several hours.
On the other hand, a method of determining a neutralizing antibody has been made known, for example, by Takashi Kitamura, "Tissue Culture Technology for Inspection of Virus", published by KINDAI SHUPPAN (1980), page 246. When an antibody against poliovirus, for instance, is determined by this method, cultivated cells originated from human being, a monkey or an ape are first inoculated with the poliovirus. (Meanwhile, the poliovirus does never grow if it is inoculated into cells originated from the sources other than human being, a monkey or an ape.) The cells inoculated with the poliovirus collapse and are deseased as the result of cytopathogenesis due to propagation of the virus. However, the reaction product of a neutralizing antibody and the virus, (the infectiousness of virus being neutralized by the neutralizing antibody), can not propagate even if it is inoculated upon a cell originated from human being or monkey so that the cell is kept to have normal form and functions. Making use of this principle, a specific virus is reacted with blood serum and then the titre of the neutralizing antibody is determined by inspecting the presence or absence, and the degree if present, of plaque and CPE (cytopathogenic effect).
However, when the poliovirus is determined by the method described in the preceding paragraph, the poliovirus must be cultivated for about 7 days in a normal test in addition to the fact that the inspection and judgement of the result should be made by a skilled person rather than being easily conducted by a person having ordinary or middle level skill. For this reason, an order of test for the determination of neutralizing antibody is not accepted even by a large scale inspection center at the present day.
On the other hand, as a method for determining antigens or antibodies in a simpler way, there has been known in the art a method wherein properties of complement component C1q binding an antigen-antibody complex is utilized. (Simpson et al.,"Jounal of Immunological Methods", Vol. 67, 167 to 172 (1984).) In this known method, glutalaldehyde or periodic acid is conjugated to the complement component C1q as a cross-linker, and peroxidase (enzyme) is conjugated via said cross-linker to the complement component C1q as a marker. Marchalonis J. J., "Biochemical Journal", Vol. 113, pp229 to 305 (1969) discloses a method in which radioactive iodine is conjugated to the complement component C1q through the chloramine T method as a marker. However, in these known methods, an enzyme or radioactive iodine is coupled with each of the complement component C1q molecules via an amino group present on the molecule generally and at random, resulting in entire modification of the molecule since the very site of each molecule having inherent properties capable of binding to an immunocomplex has been chemically modified by said cross-linker or coupler. Accordingly, the binding activity of such a marker-labelled complement component C1q for binding to an antigen-antibody complex is seriously lowered to a level not to adapt for quantitative measurement as a reagent. Moreover, a false-positive reaction takes place frequently by the latent presence of said cross-linker in the marker-labelled complement component C1q to make it impossible to continue the determination operations. It has, thus, been impossible to provide a reliable determination method for determining an antigen or antibody in a precise and reproducible manner by the use of the complement component C1q.